Organizing principles of astrocytic nanoarchitecture in the mouse cerebral cortex.

Astrocytes are increasingly understood to be important regulators of central nervous system (CNS) function in health and disease; yet, we have little quantitative understanding of their complex architecture. While broad categories of astrocytic structures are known, the discrete building blocks that compose them, along with their geometry and organizing principles, are poorly understood. Quantitative investigation of astrocytic complexity is impeded by the absence of high-resolution datasets and robust computational approaches to analyze these intricate cells. To address this, we produced four ultra-high-resolution datasets of mouse cerebral cortex using serial electron microscopy and developed astrocyte-tailored computer vision methods for accurate structural analysis. We unearthed specific anatomical building blocks, structural motifs, connectivity hubs, and hierarchical organizations of astrocytes. Furthermore, we found that astrocytes interact with discrete clusters of synapses and that astrocytic mitochondria are distributed to lie closer to larger clusters of synapses. Our findings provide a geometrically principled, quantitative understanding of astrocytic nanoarchitecture and point to an unexpected level of complexity in how astrocytes interact with CNS microanatomy.

Control of selectivity in the preparation of 2-substituted benzoazoles by adjusting the surface hydrophobicity in two solid-based sulfonic acid catalysts.

A series of metal-free tandem reactions for the synthesis of pharmaceutically important 2-substituted benzoazoles from isothiocyanates and 2-aminothiophenol under catalyst-free conditions in the presence of Et-PMO-Me-PrSO3H (1a) and SBA-15-PrSO3H (1b) as solid acids were carried out in a highly selective way under solvent free conditions. A significant selectivity changeover toward either 2-mercaptobenzoxazole or 2-aminobenzoazole derivatives could be achieved by changing the employed catalyst from the relatively hydrophobic material 1a to the more hydrophilic catalyst 1b. This simple experimental procedure with a novel selective approach toward benzoazoles accompanied by green and reusable catalysts could be considered as an alternative to the existing methods for the synthesis of 2-substituted benzoazole derivatives.

Effect of MA01 rhamnolipid on cell viability and expression of quorum-sensing (QS) genes involved in biofilm formation by methicillin-resistant Staphylococcus aureus.

A group of biosurfactants, called rhamnolipids, have been shown to have antibacterial and antibiofilm activity against multidrug-resistant bacteria. Here, we examined the effect of rhamnolipid biosurfactants extracted from Pseudomonas aeruginosa MA01 on cell growth/viability, biofilm formation, and membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) ATCC6538 bacterial cells. The results obtained from flow cytometry analysis showed that by increasing the concentration of rhamnolipid from 30 to 120 mg/mL, the cell viability decreased by about 70%, and the cell membrane permeability increased by approximately 20%. In fact, increasing rhamnolipid concentration was directly related to cell membrane permeability and inversely related to cell survival. Microtiter plate biofilm assay and laser scanning confocal microscopy analysis revealed that rhamnolipid, at a concentration of 60 mg/mL, exerts a reducing effect on the biofilm formation of Staphylococcus aureus. Real-time PCR analysis for monitoring the relative changes in the expression of agrA, agrC, icaA, and icaD genes involved in biofilm formation and related to the quorum-sensing pathway after treatment with rhamnolipid indicated a reduced expression level of these genes, as well as sortase A gene. The results of the present study deepen our knowledge regarding the use of microbial natural products as promising candidates for therapeutic applications.

Microencapsulated Multifunctionalized Graphene Oxide Equipped with Chloroquine for Efficient and Sustained siRNA Delivery.

A multifunctionalized graphene oxide (GO)-based carrier with conjugation of aminated-polyethylene glycol (PEG-diamine), octaarginine (R8), and folic acid (FA), which also contains chloroquine (CQ), a lysosomotropic agent, is introduced. The cellular uptake mechanisms and intracellular targeting of FA-functionalized nanocarriers are examined. The localized releases of CQ and siRNA intracellular delivery are evaluated. Microencapsulation of the nanocarrier complexed with genes in layer-by-layer coating of alginate microbeads is also investigated. The covalently coconjugated FA with PEG and R8 provides a stable formulation with increased cellular uptake compared to FA-free carrier. The CQ-equipped nanocarrier shows a 95% release of CQ at lysosomal pH. The localized release of the drug inside the lysosomes is verified which accelerates the cargo discharge into cytoplasm.

Patient-specific finite element analysis of heart failure and the impact of surgical intervention in pulmonary hypertension secondary to mitral valve disease.

Pulmonary hypertension (PH), a chronic and complex medical condition affecting 1% of the global population, requires clinical evaluation of right ventricular maladaptation patterns under various conditions. A particular challenge for clinicians is a proper quantitative assessment of the right ventricle (RV) owing to its intimate coupling to the left ventricle (LV). We, thus, proposed a patient-specific computational approach to simulate PH caused by left heart disease and its main adverse functional and structural effects on the whole heart. Information obtained from both prospective and retrospective studies of two patients with severe PH, a 72-year-old female and a 61-year-old male, is used to present patient-specific versions of the Living Heart Human Model (LHHM) for the pre-operative and post-operative cardiac surgery. Our findings suggest that before mitral and tricuspid valve repair, the patients were at risk of right ventricular dilatation which may progress to right ventricular failure secondary to their mitral valve disease and left ventricular dysfunction. Our analysis provides detailed evidence that mitral valve replacement and subsequent chamber pressure unloading are associated with a significant decrease in failure risk post-operatively in the context of pulmonary hypertension. In particular, right-sided strain markers, such as tricuspid annular plane systolic excursion (TAPSE) and circumferential and longitudinal strains, indicate a transition from a range representative of disease to within typical values after surgery. Furthermore, the wall stresses across the RV and the interventricular septum showed a notable decrease during the systolic phase after surgery, lessening the drive for further RV maladaptation and significantly reducing the risk of RV failure.

Comparison of Growth Performance, Pigment Synthesis, and Esterase Activity of Synechococcus sp. HS01 and Limnothrix sp. KO01 in Response to Cadmium Toxicity.

Various studies have been conducted to understand the impact of environmental pollutants on cyanobacteria due to their abundant presence in aquatic and terrestrial environments, specific morphological and physiological characteristics, and high ecological flexibility in response to environmental changes. Here, the effect of different concentrations of cadmium on two native strains of cyanobacteria, namely Synechococcus sp. HS01 and Limnothrix sp. KO01 was studied and compared with each other. In this regard, the cyanobacterial growth, pigment contents, and esterase enzyme activity were evaluated after exposure of the cells to different concentrations of cadmium (II). The toxic effects of Cd(II) on the growth rate of Limnothrix sp. KO01, even at low concentrations, tended to be higher than those for Synechococcus sp. HS01. The content of pigments decreased by an increase in Cd(II) concentration. In compliance with the cell growth, the changes occurred in pigment contents of Limnothrix sp. KO01 was more sensitive than Synechococcus sp. HS01 in the presence of different concentrations of cadmium. Flow cytometry analysis of Cd(II) effects on esterase activity of both strains after 6, 24, 48, and 72 h of exposure to Cd(II) concentrations of 9, 27, 63, and 90 µM showed that tolerance to Cd(II) toxicity in Limnothrix sp. KO01 is less than Synechococcus sp. HS01. The results obtained in this study suggest high potentials of Synechococcus sp. HS01 for heavy metal bioaccumulation due to its considerable tolerance to cadmium.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

Sex as an important factor in nanomedicine.

Nanomedicine has demonstrated substantial potential to improve the quality and efficacy of healthcare systems. Although the promise of nanomedicine to transform conventional medicine is evident, significant numbers of therapeutic nanomedicine products have failed in clinical trials. Most studies in nanomedicine have overlooked several important factors, including the significance of sex differences at various physiological levels. This report attempts to highlight the importance of sex in nanomedicine at cellular and molecular level. A more thorough consideration of sex physiology, among other critical variations (e.g., health status of individuals), would enable researchers to design and develop safer and more-efficient sex-specific diagnostic and therapeutic nanomedicine products.

Synergistic catalysis within core-shell Fe3O4@SiO2 functionalized with triethylene glycol (TEG)-imidazolium ionic liquid and tetramethylpiperidine N-oxyl (TEMPO) boosting selective aerobic oxidation of alcohols.

HYPOTHESIS: It is expected that incorporation of 2, 2, 6, 6-tetra-methyl piperidine-N-oxyl radical (TEMPO) and an imidazolium bromide bearing hydrophilic triethylene glycol (TEG) groups on Fe3O4@SiO2 core-shell may not only result in a novel highly water-dispersible/magnetically separable multi-functional catalyst system for metal-free aerobic oxidation of alcohols, which operates through a synergistic relay pathway, but it could potentially provide a strong platform for simultaneous separation and recycling of all components. EXPERIMENTS: The catalyst was prepared by anchoring TEMPO moieties onto a magnetic core-shell Fe3O4@SiO2 functionalized with an ionic liquid bearing TEG groups. The materials was characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, nitrogen adsorption-desorption isotherms, thermal gravimetric analysis, and elemental analysis. The performance of the catalyst was evaluated and quantitatively measured in the aerobic oxidation of alcohols in water. FINDINGS: The catalyst exhibited excellent and stable colloidal dispersion in water and high performance in the aerobic oxidation of various types of alcohols under metal- and halogen-free reaction conditions. As hypothesized, strong synergistic effect between functionalized components was seen in the described reaction. The catalyst displayed excellent dual-adjustable-selectivity in the oxidation of primary alcohols to either the corresponding aldehydes or carboxylic acids by tuning the reaction solvent and/or reaction time and excellent recycling behavior through a "double-separation-strategy".

Gold Nano/Micro-Islands Overcome the Molecularly Imprinted Polymer Limitations to Achieve Ultrasensitive Protein Detection.

Here, we report on an electrochemical biosensor based on core-shell structure of gold nano/micro-islands (NMIs) and electropolymerized imprinted ortho-phenylenediamine (o-PD) for detection of heart-fatty acid binding protein (H-FABP). The shape and distribution of NMIs (the core) were tuned by controlled electrodeposition of gold on a thin layer of electrochemically reduced graphene oxide (ERGO). NMIs feature a large active surface area to achieve a low detection limit (2.29 fg mL-1, a sensitivity of 1.34 × 1013 µA mM-1) and a wide linear range of detection (1 fg mL-1 to 100 ng mL-1) in PBS. Facile template H-FABP removal from the layer (the shell) in less than 1 min, high specificity against interference from myoglobin and troponin T, great stability at ambient temperature, and rapidity in detection of H-FABP (approximately 30 s) are other advantages of this biomimetic biosensor. The electrochemical measurements in human serum, human plasma, and bovine serum showed acceptable recovery (between 91.1 ± 1.7 and 112.9 ± 2.1%) in comparison with the ELISA method. Moreover, the performance of the biosensor in clinical serum showed lower detection time and limit of detection against lateral flow assay (LFA) rapid test kits, as a reference method. Ultimately, the proposed biosensor based on the core-shell structure of gold NMIs and MIP opens interesting avenues in the detection of proteins with low cost, high sensitivity and significantstability for clinical applications.

New Provisional Function of OmpA from Acinetobacter sp. Strain SA01 Based on Environmental Challenges.

An outer membrane protein A (OmpA) from Acinetobacter sp. strain SA01 was identified and characterized in-depth based on the structural and functional characteristics already known of its homologues. In silico structural studies showed that this protein can be a slow porin, binds to peptidoglycan, and exhibits emulsifying properties. Characterization of the recombinant SA01-OmpA, based on its emulsifying properties, represented its promising potentials in biotechnology. Also, the presence of SA01-OmpA in outer membrane vesicles (OMV) and biofilm showed that this protein, like its homologues in Acinetobacter baumannii, can be secreted into the extracellular environment through OMVs and play a role in the formation of biofilm. After ensuring the correct selection of the protein of interest, the role of oxidative stress induced by cell nutritional parameters (utilization of specific carbon sources) on the expression level of OmpA was carefully studied. For this purpose, the oxidative stress level of SA01 cell cultures in the presence of three nonrelevant carbon sources (sodium acetate, ethanol, and phenol) was examined under each condition. High expression of SA01-OmpA in ethanol- and phenol-fed cells with higher levels of oxidative stress than acetate suggested that oxidative stress could be a substantial factor in the regulation of SA01-OmpA expression. The significant association of SA01-OmpA expression with the levels of oxidative stress induced by cadmium and H2O2, with oxidative stress-inducing properties and lack of nutritional value, confirmed that the cells tend to harness their capacities with a possible increase in OmpA production. Collectively, this study suggests a homeostasis role for OmpA in Acinetobacter sp. SA01 under oxidative stress besides assuming many other roles hitherto attributed to this protein.IMPORTANCE Acinetobacter OmpA is known as a multifaceted protein with multiple functions, including emulsifying properties. Bioemulsifiers are surface-active compounds that can disperse hydrophobic compounds in water and help increase the bioavailability of hydrophobic hydrocarbons to be used by degrading microorganisms. In this study, an OmpA from Acinetobacter sp. SA01 was identified and introduced as an emulsifier with a higher emulsifying capacity than Pseudomonas aeruginosa rhamnolipid. We also showed that the expression of this protein is not dependent on the nutritional requirements but is more influenced by the oxidative stress caused by stressors. This finding, along with the structural role of this protein as a slow porin or its role in OMV biogenesis and biofilm formation, suggests that this protein can play an important role in maintaining cellular homeostasis under oxidative stress conditions. Altogether, the present study provides a new perspective on the functional performance of Acinetobacter OmpA, which can be used both to optimize its production as an emulsifier and a target in the treatment of multidrug-resistant strains.

Nanoscale characterization of the biomolecular corona by cryo-electron microscopy, cryo-electron tomography, and image simulation.

The biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.

Aerobic oxidation and oxidative esterification of alcohols through cooperative catalysis under metal-free conditions.

The ABNO@PMO-IL-Br material obtained by anchoring 9-azabicyclo[3.3.1]nonane-3-one N-oxyl (keto-ABNO) within the mesopores of periodic mesoporous organosilica with bridged imidazolium groups is a robust bifunctional catalyst for the metal-free aerobic oxidation of numerous primary and secondary alcohols under oxygen balloon reaction conditions. The catalyst, furthermore, can be successfully employed in the first metal-free self-esterification of primary aliphatic alcohols affording valued esters.

Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (∼8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

Aerobic Oxidation of Alcohols Catalyzed by in Situ Generated Gold Nanoparticles inside the Channels of Periodic Mesoporous Organosilica with Ionic Liquid Framework.

In situ generated gold nanoparticles inside the nanospaces of periodic mesoporous organosilica with an imidazolium framework (Au@PMO-IL) were found to be highly active, selective, and reusable catalysts for the aerobic oxidation of activated and nonactivated alcohols under mild reaction conditions. The catalyst was characterized by nitrogen adsorption-desorption measurement, thermogravimetric analysis (TGA), transmission electron microscopy (TEM), elemental analysis (EA), diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The catalyst exhibited excellent catalytic activity in the presence of either Cs2CO3 (35 °C) or K2CO3 (60 °C) as reaction bases in toluene as a reaction solvent. Under both reaction conditions, various types of alcohols (up to 35 examples) including activated benzylic, primary and secondary aliphatic, heterocyclic, and challenging cyclic aliphatic alcohols converted to the expected carbonyl compounds in good to excellent yields and selectivity. The catalyst was also recovered and reused for at least seven reaction cycles. Data from three independent leaching tests indicated that amounts of leached gold particles were negligible (<0.2 ppm). It is believed that the combination of bridged imidazolium groups and confined nanospaces of PMO-IL might be a major reason explaining the remarkable stabilization and homogeneous distribution of in situ generated gold nanoparticles, thus resulting in the highly active and recyclable catalyst system.

Palladium supported on a novel ordered mesoporous polypyrrole/carbon nanocomposite as a powerful heterogeneous catalyst for the aerobic oxidation of alcohols to carboxylic acids and ketones on water.

Preparation of an ordered mesoporous polypyrrole/carbon (PPy/OMC) composite has been described through a two-step nanocasting process using KIT-6 as a template. Characterization of the PPy/OMC nanocomposite by various analysis methods such as TEM, XRD, TGA, SEM and N2 sorption confirmed the preparation of a material with ordered mesoporous structure, uniform pore size distribution, high surface area and high stability. This nanocomposite was then used for the immobilization of palladium nanoparticles. The nanoparticles were almost uniformly distributed on the support with a narrow particle size of 20-25 nm, confirmed by various analysis methods. Performance of the Pd@PPy/OMC catalyst was evaluated in the aerobic oxidation of various primary and secondary alcohols on water as a green solvent, giving the corresponding carboxylic acids and ketones in high yields and excellent selectivity. The catalyst could also be reused for at least 10 reaction runs without losing its catalytic activity and selectivity. High catalytic efficiency of the catalyst can be attributed to a strong synergism between the PPy/OMC and that of supported Pd nanoparticles.

Nanomaterials for bone tissue regeneration: updates and future perspectives.

Joint replacement and bone reconstructive surgeries are on the rise globally. Current strategies for implants and bone regeneration are associated with poor integration and healing resulting in repeated surgeries. A multidisciplinary approach involving basic biological sciences, tissue engineering, regenerative medicine and clinical research is required to overcome this problem. Considering the nanostructured nature of bone, expertise and resources available through recent advancements in nanobiotechnology enable researchers to design and fabricate devices and drug delivery systems at the nanoscale to be more compatible with the bone tissue environment. The focus of this review is to present the recent progress made in the rationale and design of nanomaterials for tissue engineering and drug delivery relevant to bone regeneration.

A ribosomal protein S5 isoform is essential for oogenesis and interacts with distinct RNAs in Drosophila melanogaster.

In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. Here, we demonstrate that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We conclude that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.

Mouse models of hereditary hemochromatosis do not develop early liver fibrosis in response to a high fat diet.

Hepatic iron overload, a hallmark of hereditary hemochromatosis, triggers progressive liver disease. There is also increasing evidence for a pathogenic role of iron in non-alcoholic fatty liver disease (NAFLD), which may progress to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular cancer. Mouse models of hereditary hemochromatosis and NAFLD can be used to explore potential interactions between iron and lipid metabolic pathways. Hfe-/- mice, a model of moderate iron overload, were reported to develop early liver fibrosis in response to a high fat diet. However, this was not the case with Hjv-/- mice, a model of severe iron overload. These data raised the possibility that the Hfe gene may protect against liver injury independently of its iron regulatory function. Herein, we addressed this hypothesis in a comparative study utilizing wild type, Hfe-/-, Hjv-/- and double Hfe-/-Hjv-/- mice. The animals, all in C57BL/6J background, were fed with high fat diets for 14 weeks and developed hepatic steatosis, associated with iron overload. Hfe co-ablation did not sensitize steatotic Hjv-deficient mice to liver injury. Moreover, we did not observe any signs of liver inflammation or fibrosis even in single steatotic Hfe-/- mice. Ultrastructural studies revealed a reduced lipid and glycogen content in Hjv-/- hepatocytes, indicative of a metabolic defect. Interestingly, glycogen levels were restored in double Hfe-/-Hjv-/- mice, which is consistent with a metabolic function of Hfe. We conclude that hepatocellular iron excess does not aggravate diet-induced steatosis to steatohepatitis or early liver fibrosis in mouse models of hereditary hemochromatosis, irrespectively of the presence or lack of Hfe.

Development of a novel method for the purification of C-phycocyanin pigment from a local cyanobacterial strain Limnothrix sp. NS01 and evaluation of its anticancer properties.

C-phycocyanin (C-PC) pigment, as a natural blue dye, has particular applications in various fields. It is a water-soluble protein which has anticancer, antioxidant and anti-inflammatory properties. Here, we introduce an efficient procedure for the purification of C-PC pigment, followed by conducting a comprehensive investigation of its cytotoxic effects on human breast cancer (MCF-7) cells and the underlying mechanisms. A novel four-step purification procedure including the adsorption of impurities with chitosan, activated charcoal, ammonium sulfate precipitation, and ion exchange chromatography was employed, achieving a high purity form of C-PC with purity index (PI) of 5.26. SDS-PAGE analysis showed the purified C-PC with two discrete bands, subunit α (17 kD) and ß (20 kD), as confirmed its identity by Native-PAGE. A highly purified C-PC was employed to evaluate its anticancer activity and underlying molecular mechanisms of action. The inhibitory effects of highly purified C-PC on the proliferation of human breast cancer cells (MCF-7) have detected by MTT assay. The IC50 values for 24, 48, and 72 hours of exposure to C-PC were determined to be 5.92, 5.66, and 4.52 µg/µl, respectively. Flow cytometric analysis of cells treated with C-PC, by Annexin V/PI double staining, demonstrated to induce MCF-7 cells apoptosis. Also, the results obtained from propidium iodide (PI) staining showed that MCF-7 cells treated with 5.92 µg/µl C-PC for 24 h would arrest at the G2 phase and 5.66 and 4.52 µg/µl C-PC for 48 and 72 h could induce cell cycle arrest at both G2 and S phases. The oxidative damage and mitochondrial dysfunction were evaluated to determine the possible pathways involved in C-PC-induced apoptosis in MCF-7 cells. Our findings clearly indicated that the treatment of MCF-7 cells with C-PC (IC50 for 24 h) increased the production of reactive oxygen species (ROS). Consequently, an increase in the lipid peroxidation (LPO) level and a reduction in the ATP level, mitochondrial membrane potential (MMP), glutathione (GSH) and its oxidized form (GSSG), occurred over time. The reduced expression levels of anti-apoptotic proteins, Bcl2 and Stat3, plus cell cycle regulator protein, Cyclin D1, using Real-Time PCR confirm that the C-PC-induced death of MCF-7 human breast cancer cells occurred through the mitochondrial pathway of apoptosis. Collectively, the analyses presented here suggest that C-PC has the potential so that to develop it as a chemotherapeutic anticancer drug.